Regenerative potential of ASC-derived conditioned medium (CM)

OBJECTIVE: Adipose-derived Stem Cells (ASCs) are multipotent progenitors able to participate in damaged tissue regeneration and to modulate immune response. Their regenerative and protective action might be mainly due to the release of soluble factors and/or extracellular vesicles (EVs) which might mediate immunomodulation, proliferation and differentiation of the surrounding cells. We have found that hASC-conditioned medium (CM) counteracts neuropathic pain symptoms in preclinical mouse models. Here we show the ASC secretome effects in vitro on primary cells and the biophysical characterization of its EVs. MATERIALS AND METHODS: ASC were isolated from subcutaneous adipose tissue and cultured in cDMEM. After a 72-hours-starving period, ASC CM was collected, concentrated through Amicon Ultra-15 Centrifugal Filter Unit (Merck-Millipore) and its protein content quantified by Bradford assay. The trophic effect was tested on terminally differentiated cells (human osteoblasts, OBs, and human chondrocytes, CHs) and progenitors (ASCs) by Alamar Blue and MTT assays. Micro-vesicles (MVs) of 500-1000 nm and MVs-depleted CM (CM-MVs) were fractionated by centrifugation at 10,000xg, while exosomes (80-100 nm) and smaller micro-vesicles (<500 nm) were isolated after centrifugation at 100,000xg. EV-cell interaction has been evaluated culturing hOB up to 10 days with CM from PKH26-stained ASCs and looking for EVs incorporation. Finally, the composition of ASC derived-EVs was determined by Raman spectroscopy. RESULTS: Our ASC-CM was concentrated 45±10-folds (n=15) and its protein content was 53.5±27.0 µg /106 cells (n=12). 7 days of culture with both CM and CM-MVs improved viability of both OBs and ASCs (+ about 70%), and CHs (more than 100%). In contrast, MVs alone had no effect, although they are easily and markedly incorporated into OBs-plasma membrane after 4 days of treatment. Raman analysis provided a biochemical overview of EVs isolated from ASCs-derived CM which were also different from vesicles derived from other cell types. CONCLUSION: Concentrated ASCs-derived CM represents a powerful source of factors with future therapeutic potential. We can standardize the production of CMs so their effects can be more easily compared in the optic of a clinical grade production. Our in vitro data show a trophic action of CM, and that ASC-released vesicles interact with recipient cells, most likely releasing their content. We suggest that soluble factors in the CM or in the exosomes, rather than in the bigger MVs, might be involved in the ASC trophic anti-inflammatory potential.